Wilhelm H. Schmitt

Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo

Apart from the diagnostic value of anti‐neutrophil cytoplasmic antibodies (ANCA), their detailed characterisation and that of their corresponding antigens have opened new ways for the exploration of the pathogenesis of primary systemic vasculitis. ANCA arc now thought to play an important functional role via activation of phagocytic cells (e.g. polymorphonuclear neutrophils (PMN)). In this study we examined the mechanisms by which ANCA could gain access to proteinase 3 (PR3) in intact PMN, at two levels: ex vivo by analysing the presence of PR3 on the plasma membrane of PMN from patients with ANCA‐associated vasculitis, and in vitro by stimulation of PMN using different cytokines, including recombinant tumour necrosis factor‐alpha (rhTNF‐α) and two forms of IL‐8 (produced by monocytic and endothelial cells). Using immunocytochemical staining techniques (FACS and immunoelectronmicroscopy) PR3 has been detected on the plasma membrane of PMN from patients with active ANCA‐associated vasculitis. However, this phenomenon is also seen in patients with sepsis who do not have ANCA. In addition. TNF‐α and both forms of IL‐8 act synergistically and induce a translocation of PR3 from the intragranular loci to the cell surface of PMN. These results provide strong evidence for the hypothesis that ANCA are directly pathogenic by binding to PR3 which is expressed on the cell surface of primed/activated PMN.

Churg-Strauss syndrome serum markers of lymphocyte activation and endothelial damage

OBJECTIVE To find serologic markers of disease activity in patients with Churg-Strauss syndrome (CSS) linked to possible pathogenetic mechanisms by studying endothelial cell damage (soluble thrombomodulin [sTM]) in relation to T cell and eosinophil activation markers (soluble interleukin-2 receptor [sIL-2R] and eosinophil cationic protein [ECP]), and the presence of autoantibodies (antineutrophil cytoplasmic antibodies [ANCA] and anti-endothelial cell antibodies [AECA]) during both active and inactive phases of disease. METHODS Sixteen consecutive patients who fulfilled the 1992 Chapel Hill definition of CSS were studied over a period of 4.5 +/- 3.9 years (mean +/- SD). ECP was detected by Columbo immunocapture (immunoCAP) assay, sIL-2R and sTM by enzyme-linked immunosorbent assay (ELISA), AECA by cell ELISA, and ANCA by indirect immunofluorescence and ELISA. RESULTS In patients with active disease, ECP (8.4 +/- 90 units/ml), sIL-2R (3,725 +/- 2,310 units/ml), and sTM levels (5.5 +/- 2.9 units/liter) were significantly elevated compared with those in remission. Levels of sIL-2R showed a close correlation with levels of sTM (r = 0.75, P < 0.05). Interestingly, during remission, sIL-2R levels remained elevated in 4 of 7 patients, although the erythrocyte sedimentation rate, C-reactive protein level, and sTM level returned to the normal range (levels > 1,000 units/ml were associated with relapse). ANCA were found in only 7 patients (4 had classic ANCA, 3 had perinuclear ANCA), and AECA in 11 sera from 8 patients. In contrast to AECA, ANCA were associated with active disease. CONCLUSION In its active state, CSS is associated with markedly increased levels of sIL-2R and ECP, indicating T cell and eosinophil activation. Elevated sTM is a sign of endothelial cell damage that can be closely linked to T cell activation, as indicated by increased sIL-2R levels.

Clinical relevance of elevated serum thrombomodulin and soluble E-selectin in patients with Wegener's granulomatosis and other systemic vasculitides

PURPOSE Vascular injury plays an important pathophysiological role in vasculitis. Soluble serum thrombomodulin (sTM), a promising marker of endothelial cell injury, is released into the circulating blood following cell damage and was therefore investigated as a parameter of disease activity in patients with Wegeners granulomatosis (WG) and various other vasculitides. PATIENTS AND METHODS One hundred and ninety-seven sera of 102 patients with histologically proven WG of different disease activity and 41 sera of patients with other vasculitides at their active stage were investigated (12 Takayasu arteritis [TA], 7 giant cell arteritis [GCA], 10 polyarteritis nodosa [PAN], 12 Behcets disease [BD]). The sera were examined for the levels of sTM and sE-selectin (CD62E) by enzyme-linked immunosorbent assay (ELISA) and for the presence of classical anti-neutrophil cytoplasmic antibodies (cANCA) by indirect immunofluorescence (IIF). The disease activity was evaluated according to the clinical symptoms and organ involvement. RESULTS A significant increase of sTM levels compared with control values (26 +/- 2 ng/ml) was found in active WG, TA, GCA, PAN, and BD: limited active WG: 63 +/- ng/ml; generalized active WG: 119 +/- 15 ng/ml; limited WG, partial remission: 60 +/- 5 ng/ml; generalized WG, partial remission: 75 +/- 7 ng/ml; active TA: 36 +/-; active GCA: 36 +/- 4 ng/ml, active PAN: 33 +/- 2 ng/ml, active BD: 40 +/- 4 ng/ml. Limited and generalized WG in complete remission did not have elevated levels of sTM. sTM values closely reflected relapses and therapy-induced remissions of WG. Elevated cANCA titers were correlated as well with the disease activity in WG but more weakly than sTM levels. In contrast, sE-selectin values were not significantly correlated with the disease activity and the course of disease. CONCLUSIONS sTM is a promising serological marker of disease activity and progression in active limited and generalized WG and other vasculitides reflecting the degree of endothelial cell damage. sTM might prove to be a clinically useful marker for therapeutic considerations.

ANCA in Systemic Vasculitides, Collagen Vascular Diseases, Rheumatic Disorders and Inflammatory Bowel Diseases

It was the aim of this study to reevaluate the diagnostic significance and clinical implication of ANCA after testing sera from 13,606 patients for the presence of ANCA. Our data confirm the high specificity (97%) and sensitivity (80%) of cANCA for Wegeners granulomatosis. pANCA were found in renal vasculitides (60%), collagen vascular diseases (SLE 20%, Sjögrens syndrome 26%, polymyositis 16%) and rheumatic disorders (Feltys syndrome 50%, rheumatoid arthritis 20%). A third fluorescence pattern in sera of patients with inflammatory bowel disease (ulcerative colitis 28/72, Crohns disease 6/84), here called xANCA, was seen. Target antigens of granule proteins from PMN and monocytes (proteinase 3, myeloperoxidase, elastase, cathepsin G, lactoferrin, lysozyme) were identified.

Autoantibodies Directed Against Lysozyme: A New Target Antigen for Anti-Neutrophil Cytoplasmic Antibodies (ANCA)

ANCA-positive sera from 1138 patients and ANCA-negative sera from 90 patients were screened for autoantibodies directed against lysozyme (LZ) by ELISA. Sera from 120 patients did react with LZ. 99 sera bound to LZ only, whereas 56 sera bound to further granule proteins, especially cathepsin G and lactoferrin. In the routine ANCA screening, most of the anti-LZ-positive sera showed a pANCA fluorescence. In total, 8% of 674 pANCA-positive sera did react with LZ. Clinically, anti-LZ antibodies were associated inflammatory rheumatologic, -renal and -bowel diseases.

ANTINEUTROPHIL CYTOPLASMIC AUTOANTIBODIES : IMMUNOBIOLOGICAL ASPECTS

SummaryAntineutrophil cytoplasmic autoantibodies (ANCA) specific for constituents of neutrophil primary granules and monocyte lysosomes have been demonstrated in various vasculitic disorders. The staining pattern in indirect immunofluorescence microscopy using alcohol-fixed neutrophils as substrate allows distinction among 3 types of ANCA: 1) classic anti-neutrophil cytoplasmic antibody (cANCA, formerly known as ACPA); 2) a type with aperinuclear/nuclear staining pattern produced when alcohol-fixed neutrophils are used as substrate (pANCA); and 3) a mixture of both of the above types (xANCA, also described recently as pANCA). Most cANCA are directed against proteinase 3 (“Wegeners autoantigen”). Some pANCA have specificity for myeloperoxidase and are associated with idiopathic crescentic glomerulonephritis (“renal vasculitis”) and other systemic vasculitides exhibiting a paucity of immune deposits in blood vessels. In addition to being a useful serological marker, ANCA appear to be directly involved in the pathogenesis of systemic vasculitis. ANCA can activate cytokine-primed granulocytes and monocytes to undergo a respiratory burst and degranulation. This effect leads to vasculitis through the attachment of these cells to the vascular endothelium primed by cytokine-induced expression of adhesion molecules (E-LAM 1) on the endothelium. Thus, the release of toxic oxygen radicals and lytic enzymes is capable of causing vascular damage. In the present paper we report on the main target antigens and on the history, nomenclature, laboratory methods, and ethiopathological implication of ANCA. Additional pathophysiological aspects of ANCA and/or autoreactive T cells and immmunoregulatory events are also discussed.

Membrane Surface Proteinase 3 Expression and Intracytoplasmic Immunoglobulin on Neutrophils from Patients with ANCA-Associated Vasculitides

We studied the presence of proteinase 3 (PR3), myeloperoxidase (MPO) and elastase (HLE) on the plasma membrane of neutrophils in patients with biopsy-proven Wegeners disease (WG), pANCA-positive vasculitis, control patients (SLE, rheumatoid arthritis, ankylosing spondylitis), sepsis patients and healthy donors. We found an overexpression of PR3 on the cell surface of neutrophils in WG, ANCA-associated vasculitis and during infection (sepsis). Thus PR3 becomes accessible to ANCA. Furthermore we detected intracytoplasmic IgG antibodies in PMN from patients with WG by immunoelectron microscopy and direct immunofluorescence. Our findings support the pathophysiological role of ANCA.

Use of Highly Sensitive C-Reactive Protein for Followup of Wegener’s Granulomatosis

Objective. Since Wegener’s granulomatosis (WG) represents a relapsing disease, efforts have been made to reliably predict relapses using blood tests. Followup measures such as conventionally determined C-reactive protein (CRP), antineutrophil cytoplasmic antibody (C-ANCA) titer, and proteinase-3 (PR3) ELISA are applied. We evaluated whether during remission elevated highly sensitive CRP (hsCRP) precedes relapse as a marker of subclinical inflammation and thus might improve clinical assessment. Methods. We investigated 227 sera of 57 patients with WG: 74 sera collected from patients in remission who subsequently relapsed (before relapse), 30 sera collected during relapse, and 123 sera from patients in remission without relapse. We also distinguished between major and minor relapse. hsCRP, conventionally determined CRP (CRP), C-ANCA, PR3-ELISA, and erythrocyte sedimentation rate (ESR) were measured using commercial kits, and levels were correlated to clinical status. Results. Only hsCRP and ANCA titer, but not CRP levels, were higher in sera from patients who subsequently relapsed versus those who did not, indicating patients at risk. Levels of hsCRP, CRP, and ESR were higher in sera collected during relapse than in the sera before relapse. hsCRP, conventional CRP, and ESR were also higher in samples collected during major relapse than before major relapse. Looking at the levels just before relapse compared to previous levels during remission, none of these measures rose directly before the clinical manifestation of the relapse. Conclusion. Our study provides evidence for an additional value of hsCRP in the clinical assessment of patients with WG.

Differentiation Between Wegener’s Granulomatosis and Microscopic Polyangiitis by an Artificial Neural Network and by Traditional Methods

Objective. To investigate the operating characteristics of the American College of Rheumatology (ACR) traditional format criteria for Wegener’s granulomatosis (WG), the Sørensen criteria for WG and microscopic polyangiitis (MPA), and the Chapel Hill nomenclature for WG and MPA. Further, to develop and validate improved criteria for distinguishing WG from MPA by an artificial neural network (ANN) and by traditional approaches [classification tree (CT), logistic regression (LR)]. Methods. All criteria were applied to 240 patients with WG and 78 patients with MPA recruited by a multicenter study. To generate new classification criteria (ANN, CT, LR), 23 clinical measurements were assessed. Validation was performed by applying the same approaches to an independent monocenter cohort of 46 patients with WG and 21 patients with MPA. Results. A total of 70.8% of the patients with WG and 7.7% of the patients with MPA from the multicenter cohort fulfilled the ACR criteria for WG (accuracy 76.1%). The accuracy of the Chapel Hill criteria for WG and MPA was only 35.0% and 55.3% (Sørensen criteria: 67.2% and 92.4%). In contrast, the ANN and CT achieved an accuracy of 94.3%, based on 4 measurements (involvement of nose, sinus, ear, and pulmonary nodules), all associated with WG. LR led to an accuracy of 92.8%. Inclusion of antineutrophil cytoplasmic antibodies did not improve the allocation. Validation of methods resulted in accuracy of 91.0% (ANN and CT) and 88.1% (LR). Conclusion. The ACR, Sørensen, and Chapel Hill criteria did not reliably separate WG from MPA. In contrast, an appropriately trained ANN and a CT differentiated between these disorders and performed better than LR.

Effectiveness of Cyclophosphamide Pulse Treatment in Wegener’s Granulomatosis

43 patients with Wegener’s granulomatosis were studied to evaluate the effectiveness of cyclophosphamide (cyc) pulse treatment. 42% of the patients showed benefits from treatment for at least 6 months after the cessation of cyc pulses. Analysis of clinical and laboratory parameters indicate that this treatment is less effective in patients in whom more than 4 organ systems are involved. Responders to the treatment showed disease manifestations predominantly in the ENT and lower respiratory tract and had lower cANCA titers ( < 1:64) prior to treatment.